1. Technical Field
The present invention relates to a new class of chemiluminescent, aromatic ring-fused acridinium compounds (AFAC) which emit green or yellow light upon simple chemical treatments. This invention also relates to conjugates formed from AFAC and binding partners, e.g. biological molecules, and test assays utilizing the conjugates. Furthermore the invention relates to test assays in which the detection and/or quantitation of two or more substances or analytes in a test sample can be carried out simultaneously due to the discernable and non-interfering light emission characteristics of two or more chemiluminescent conjugates.
2. Cross-Reference
The following application, filed concurrently herewith, describes a luminometer for detecting emission spectra of at least two chemiluminescent compounds in a test sample and is identified as U.S. Ser. No. 08/035,341. The disclosure of said application is commonly assigned and incorporated herein by reference.
3. Technical Review
Chemiluminescent compounds that emit light with separated wavelength maxima and minimally overlapping but correctable emission spectra can be very useful in analytical assays, particularly in industrial assays, and in clinical diagnostic assays for multi-substance, e.g. multi-analyte, determinations. Such compounds can be used to tag or label binding partners, e.g. biological molecules, such as antigens, antibodies, and nucleic acids to form conjugates or tracers that are capable of producing mutually non-interfering, or minimally over-lapping light emission signals or spectra, that allow the simultaneous detection and/or quantitation of multiple substances in a test sample. For example, simultaneous determinations of serum levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from one patient sample is possible and is demonstrated below, because two chemiluminescent compounds having light emission spectra which span about 100-250 nm for a spectral region with signal intensity above 5% of peak height, but differing in their emission maxima so that the signals are discernable. In one example the emission maxim of two chemiluminescent signals differ by about 60 nm and preferably by 80 nm or more for labeling or tagging anti-LH and anti-FSH. A further example where simultaneous determinations is possible according to the methods described herein, is in the assay of amplified nucleic acid sequences, e.g. oncogenes associated with malignant transformation. See EP-A-0 481 704 (priority app. U.S. Ser. No. 598,269 (Oct. 16, 1990) and references cited therein) which is commonly assigned and incorporated herein by reference. In such assays, the inclusion of a parallel, internal reference material for a known, different target sequence in the same working vessel or reaction medium as a positive control is recognized as important to assay performance, for example, to safeguard false negative results. In other assay formats, the inclusion of a known control substance will also serve to assess assay performance.
The economical benefit and the experimental necessity of determining and/or quantitating two or more substances, e.g. analytes, in a test sample were the two main underlying motives in the development of a multiple-tracer assay system of the present invention. It was further envisioned that an ideal multiple-tracer system would emit multiple-wavelength signals under identical chemical conditions. It was recognized that it would be less desirable and more cumbersome to combine two chemiluminescent tracers in a multiple-analyte assay system that required two different sets of signal generating mechanisms, conditions and timings as would be in the case of utilizing two different classes of chemiluminescent compounds such as acridinium compounds pairing with luminol series or with stable dioxetanes, which involve the use of other chemicals or enzymes to generate the signal. Furthermore the two or more different chemiluminescent compounds or conjugates must have emission efficiency differing by not more than one order of magnitude.
A still further fundamental requirement was the adequate stability of the chemiluminescent compounds in aqueous media or environment, which will withstand the shipping conditions for commercial products when placed in kit form.
Applicant has achieved these goals by developing stable chemiluminescent analogues within the same general class that exhibit bathochromic shifts in their emission maxima, and emit light with comparable efficiency under identical chemical treatments.
Chemiluminescent acridinium compounds which are shown herein to emit blue light upon treatment with hydrogen peroxide and metal hydroxide have been well documented. U.S. Pat. Nos. 4,745,181, 4,918,192, 5,110,932, U.S. Ser. No. 07/826,186, U.S. Pat. No. 5,227,489, and U.S. Ser. No. 07/871,601, U.S. Pat. No. 5,241,070, describe stable polysubstituted-aryl acridinium esters; all of which are commonly assigned and incorporated herein by reference. Such acridinium compounds shall be referred to generally herein as "reference acridinium esters" or "acridinium esters". Such compounds as indicated include an acridinium ring system and, depending on the use of such compounds, further include an appropriate functional group(s), e.g. for attaching the label to a substance to form conjugates for use in a test assay, including the assays of the present invention.
Batmanghelich, et al, EP-A-0 478 626 (priority GB 2233450A (Jun. 24, 1989)), described the use of acridinium compounds of varying light emission, i.e. fast and slow durations to prepare different tracer conjugates, to achieve a "substantially simultaneous" quantitation of two or more different analytes. This approach, however, has several major drawbacks. First, one of the acridinium esters includes electron-withdrawing substituents on the phenolic moiety in order to achieve very short duration of light emission, i.e. complete emission or emission maxima in one second. This type of compound, e.g. a ortho-dihalogenated aryl acridinium ester, however, may suffer from lack of stability in aqueous environment. Second, light emission kinetics must be carefully examined to permit accurate correction in order to distinguish the light emission contributed individually by the two tracers during the overlapping period of light emission. The described method relies on the measurement of photons emitted in two separate time windows for sequential integration of light intensity. Unless the light emission overlap is relatively small, such correction could be a potential source for poor assay precision, particularly for detection of two analytes having widely different concentrations. The requirement for smaller light emission overlap would in turn demand the availability of a pair of chemiluminescent compounds, one having very short and the other very long duration of light emission; and would lead to compounds which either have stability problems or render the dual-analyte assay unpractical due to excessively prolonged signal-collection time.
Where the signal collection time is extended, an advantage of performing two assays in a single test sample would be lost. It is noted that in one automated analyzer using chemiluminescent detection and/or quantitation, a first test result is reported at fifteen (15) minutes and thereafter at every twenty (20) seconds during operation, see EP-A-0 502 638 (priority U.S. Ser. No. 665,196 (Mar. 4, 1991, abandoned)) which is commonly assigned and incorporated herein by reference.
Batmanghelich et al also described an acridinium compound of different light emission spectra to prepare different tracer conjugates. The approach they used was to extend the electronic conjugation of the acridinium nucleus to obtain 3-(4-carboxybutadienyl)-acridinium ester (compound 2b) with bathochromic shift of about 80 nm in the emission maximum as compared to the parent acridinium ester (compound 2a). Extension of electronic conjugation of the acridinium nucleus does not necessarily lead to major bathochromic shift in the emission maximum which is practically needed to construct a dual-analyte immunoassays and a possible reduction in emission efficiency. No teaching was made of benzacridinium chemiluminescent compounds or conjugates for use in such assays.
McCapra et al, EP-A-0 322 926 (priority U.S. Ser. Nos. 140,040 (Dec. 31, 1987), abandoned, and 291,843 (Dec. 29, 1988), abandoned), suggested the "chemiluminescent moiety" consisting of heterocyclic ring or ring system with ester, amide linkages attached to one of the carbon atoms on the ring or ring system. This chemiluminescent compound was said to include benz[a]acridinium, benz[b]acridinium, and benz[c]acridinium but the synthesis and structure of these compounds or conjugates was not described. Neither emission wavelength maxima, nor light emission efficiency of these structures were predicted; nor were the use of at least two chemiluminescent compounds or conjugates in an assay method, nor the utility of such compounds when used in assays based on their emission spectra.
The nomenclature of benz[a]acridinium and benz[b]acridinium utilized in this disclosure is based on Rule 21.5 of Definitive Rules for Nomenclature of Organic Chemistry, Ed. International Union of Pure and Applied Chemistry in the 1957 REPORT OF THE COMMISSION ON THE NOMENCLATURE OF ORGANIC CHEMISTRY.
According to the example given on Benz[a]anthracene, the compound arising from fusing benzene ring to the peripheral sides of the acridnium nucleus (structure below) should therefore be named according to whether side a, b, or c of the acridinium nucleus is fused with the benzene ring. ##STR1##
The following abbreviations are utilized in the disclosure:
______________________________________ 1. ABAC: angular benz[a]acridinium compound 2. AFAC: aromatic ring fused acridinium compound 3. EtO: ethoxy 4. DMAE: dimethyl acridinium ester 5. DIPAE: diisopropyl acridinium ester 6. LBAC: linear benz[b]acridinium compound 7. LEAC: longer emission acridinium compound 8. LEAE: longer emission acridinium ester 9. MeO: methoxy 10. NSE: N-sulfoethyl 11. NSP: N-sulfopropyl 12. PCT: percent cross talk 13. PMP: paramagnetic part icles 14. QAE: quaternary ammonium ethoxy 15. RLU: relative light units ______________________________________